Beard et al. Parasites Vectors (2021) 14:60 https://doi.org/10.1186/s13071-020-04565-6 Parasites & Vectors

RESEARCH Open Access Morphological identifcation of and molecular detection of ‑borne from bare‑nosed wombats (Vombatus ursinus) Danielle Beard1, Hayley J. Stannard2 and Julie M. Old1*

Abstract Background: Ticks are obligate haematophagous ectoparasites of vertebrate hosts and transmit the widest range of pathogenic organisms of any . Seven tick species are known to feed on bare-nosed wombats (Vom- batus ursinus), in addition to the highly prevalent scabiei which causes fatal sarcoptic mange in most bare-nosed wombat populations. Little is known about the pathogens carried by most wombat ticks or how they may impact wombats and wombat handlers. Methods: Wombat ticks were sourced from wildlife hospitals and sanctuaries across and identifed to spe- cies level using taxonomic keys. Genomic DNA was extracted from a subsample, and following the amplifcation of the bacterial 16S rRNA gene V3–V4 hypervariable region, next-generation sequencing (NGS) on the Illumina MiSeq platform was used to assess the microbial composition. Results: A total of 447 tick specimens were collected from 47 bare-nosed wombats between January 2019 and Janu- ary 2020. Five species of ticks were identifed comprising wombat tick Bothriocroton auruginans (n 420), wallaby tick bancrofti (n 8), bush tick Haemaphysalis longicornis (n 3), common marsupial tick= tasmani (n 12), and Australian paralysis= tick (n 4). Tick infestations= ranged from one to 73 ticks per wombat. The= wombat tick was the most prevalent tick species comprising= 94% of the total number of samples and was pre- sent on 97.9% (46/47) of wombat hosts. NGS results revealed the 16S rRNA gene diversity profle was predominantly (55.1%) followed by (21.9%) and Actinobacteria (18.4%). A species of Coxiella sharing closest sequence identity to (99.07%), was detected in 72% of B. auruginans and a Rickettsiella dominated the bacterial profle for I. tasmani. Conclusions: A new host record for H. longicornis is the bare-nosed wombat. One adult male and two engorged adult female specimens were found on an adult male wombat from Coolagolite in New South Wales, and more specimens should be collected to confrm this host record. The most prevalent tick found on bare-nosed wombats was B. auruginans, confrming previous records. Analysis of alpha-diversity showed high variability across both sample locations and instars, similar to previous studies. The detection of various Proteobacteria in this study highlights the high bacterial diversity in native Australian ticks. Keywords: Wombat, Tick, Microbiome, Marsupial, 16S ribosomal RNA gene, Next-generation sequencing,

*Correspondence: [email protected] 1 School of Science, Western Sydney University, Penrith, New South Wales, Australia Full list of author information is available at the end of the article

© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco​ ​ mmons.org/licen​ ses/by/4.0/​ . The Creative Commons Public Domain Dedication waiver (http://creativeco​ mmons​ .org/publi​ cdoma​ in/​ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Beard et al. Parasites Vectors (2021) 14:60 Page 2 of 18

Background with native tick species, and healthy wombats regularly Ticks (: ) are obligate ectoparasitic arach- carry large burdens of ticks which would otherwise afect nids that are classifed into three families: Ixodidae (hard humans and domestic [20]. However, wombats ticks), (soft ticks), and Nuttalliellidae. Each of afected by sarcoptic mange, orphaned or injured wom- the three families have evolved unique biological, physi- bats released from captivity and wombats raised in a ological and ecological disparities which have resulted comparatively parasite-free captive environment before in diferent abilities and capacities to transmit patho- release are likely at an increased risk of contracting tick- gens [1]. However, ticks can transmit the widest range of borne diseases. Managing wild species in captivity may pathogens of any arthropod vector and are the primary induce stress, impair immunity and expose hosts to novel cause of vector-borne diseases in and domestic parasites to which the immune system is naïve [21]. Pop- animals [2]. Ixodids transmit the widest number of path- ulation density is also often atypical in captivity, which ogens worldwide and are responsible for the majority of may result in higher than usual parasite burdens. Addi- tick-borne [3]. tionally, the use of anti-parasitic medications on captive In addition to pathogens, the tick microbiome com- animals afects both host-parasite relationships and indi- prises a community of commensal and symbiotic obli- viduals, as the latter are at an increased risk of disease gate which make up the majority of the once released, having not developed acquired immunity tick microbiome and reside both inside and outside the [22]. body of ticks [4]. Te efect of these organisms has been Seven species of ticks have previously been recorded somewhat neglected in studies, but may present various feeding on bare-nosed wombats including the wom- detrimental, neutral, or benefcial efects to their tick bat tick Bothriocroton auruginans [23][23], wallaby tick hosts, and also contribute to driving the of Haemaphysalis bancrofti [24], Australian paralysis tick tick-borne pathogens [5]. Non-pathogenic microorgan- Ixodes holocyclus [25], Tasmanian paralysis tick Ixodes isms are typically transovarially transmitted [6] and may cornuatus [26], Ixodes phascolomyis [27], common mar- impact tick growth, reproduction, ftness, nutritive adap- supial tick Ixodes tasmani and Ixodes victoriensis [28] tation and defence against environmental stresses [7, 8]. [28]. Te relationship between S. scabiei and other Te functional roles of tick microorganisms and their known wombat ectoparasites, their pathogens, ability to relationships may provide further insights into the path- co-infect hosts, and their overall impact on wombat hosts ogenicity and evolution of tick pathogens. For example, have not yet been investigated. Tere is also very little it has become increasingly clear since the advancement known about the life cycles of wombat ectoparasites and of molecular barcoding techniques that many species of their level of host specifcity. Coxiella burnetii has been , Francisella, and Coxiella, which are generally found in B. auruginans collected from bare-nosed wom- considered pathogens of medical and veterinary impor- bats, as well as a Rickettsia species closely related to Rick- tance, have evolved as non-pathogenic endosymbionts of ettsia massiliae, which causes human disease [29]. Tese ticks [9]. are the only pathogens that have been detected in ticks While tick-borne bacteria have been relatively well taken from wombat hosts, and were identifed using spe- studied in the northern hemisphere, very little is known cifc targeted methods. about the presence or diversity of bacteria in Australian Te development of next-generation sequencing (NGS) ticks [10]. Te microbiome and pathogenicity of Austral- technologies has enabled the microbial communities ian ticks are unique when compared to other species, and of ticks to be explored in a fast and cost-efcient man- so is the response to ticks and tick-borne pathogens from ner [15]; however, very little is known about the micro- native vertebrate hosts [11]. Recently, unique Australian biome of native Australian ticks [10] and no studies species of Anaplasma, Ehrlichia and Neoehrlichia [12, have focused on wombat ticks or tick-borne pathogens. 13] and the frst native species were character- Bare-nosed wombats are already signifcantly afected ised in native ticks [14]. Other novel microbial species by a known ectoparasite, so it would be benefcial to have also been reported in Australian ticks [12, 15, 16]; understand the other parasitic and pathogenic threats however, the focus has largely been on ticks of human, that wombats may need to overcome simultaneous to domestic and livestock importance, and few stud- or following the treatment of sarcoptic mange. It is also ies have surveyed ticks associated with wildlife [17, 18]. important to identify potential zoonotic threats to wom- Bare-nosed wombat (Vombatus ursinus) populations bat handlers and domestic animals that may come into are signifcantly impacted by the ectoparasite Sarcoptes contact with wombats or their burrows. Tis study aimed scabiei, which causes sarcoptic mange [19]; however, lit- to identify the species of ticks associated with bare-nosed tle is known about other wombat ectoparasites or their wombats and to use NGS and metabarcoding to investi- associated pathogens. Australian fauna have co-evolved gate the bacterial diversity associated with these ticks. Beard et al. Parasites Vectors (2021) 14:60 Page 3 of 18

Methods the V3-V4 region of the 16S rRNA gene, generating a Tick collection and identifcation 300-base pair fragment. All ticks were collected directly from wombat hosts Te bioinformatics analysis involved demultiplex- between January 2019 and January 2020 throughout east- ing, quality control, operational taxonomic unit (OTU) ern Australia (Fig. 1) including from live animals being clustering, and taxonomic classifcation. Image analy- rehabilitated for release, as well as opportunistic collec- sis was performed in real time using MiSeq Control tions from road-killed wombats, and placed into 70% Software version 2.6.2.1 and Real Time Analysis ver- ethanol. Te location where the ticks were collected, sion 1.18.54 (Illumina, San Diego, CA), running on the date, and habitat type for the wombat hosts were the instrument computer. Ten the Illumina bcl2fastq recorded. Temperature and rainfall were obtained from 2.20.0.422 pipeline was used to generate the sequence the Bureau of Meteorology for the date and Global Posi- data. Paired-ends reads were assembled by aligning tioning System coordinates where ticks were submitted. the forward and reverse reads using PEAR version All ticks were identifed morphologically to species and 0.9.5 [38], and primers were identifed and trimmed. life stage using existing taxonomic keys [30, 31] and a Trimmed sequences were processed using Quantitative Nikon SMZ445 stereomicroscope. Species, sex and instar Insights into Microbial Ecology (QIIME) version 1.8.4 were recorded for each specimen except for two nymphal [39], USEARCH version 8.0.1623 [40], and UPARSE specimens and specimens that were damaged during [41] software. Using USEARCH tools, sequences removal. Tere is a lack of detailed morphological keys were then quality fltered, full-length duplicates were for some Australian native ticks at the larval and nym- removed and sequences were sorted by abundance. phal stages [32], so some of these specimens could only Singletons or unique reads were discarded, sequences be identifed to genus level. Damaged ticks were identi- were clustered and chimeric sequences were fltered fed to instar and genus. Photographs of tick specimens using the rdp_gold database as a reference. To obtain were taken using an Olympus DP72 stereomicroscope the number of reads in each OTU, reads were mapped with an external Euromex EK-1 fbre optic light source back to OTUs with a minimum identity of 97%, taxon- and cellSens Standard version 1.5 software. Ticks were omy was assigned using QIIME and taxonomies were stored in sterile tubes containing 70% ethanol between confrmed using the National Center for Biotechnology identifcation and molecular analysis. Information MegaBLAST. Non-bacterial (eukaryote, unidentifed) OTUs were removed and samples with Sample mapping <100 assigned OTUs were not considered a positive Te locations of tick sample collection were geo-ref- identifcation. erenced using the open source software QGIS version 3.12.1 [33] with the latest Australian coordinate sys- Data management and statistical analyses tem Geocentric Datum of Australia 2020 incorporated Tick collection and identifcation details were recorded through the ICSM NTv2 Transformer plugin [34]. Layers in Microsoft Excel version 2002. Quality assurance was were styled with a categorised renderer and layer symbol- ensured prior to statistical analyses by reviewing all ogy was characterised according to tick species. To visu- physical data and data entries. Statistical analyses and alise overlapping points, a point displacement renderer data visualization were performed using RCommander was used around a centre symbol on rendering circles for version 2.6-2 [42], RStudio version 1.2.5033 [43] with the tick distribution, and a point cluster renderer was used to addition of packages vegan version 2.5-6 [44] and phy- visualise overlying distribution [35]. loseq [45], and Geneious Prime 2020.1.1 (https​://www. genei​ous.com). Alpha-diversity was assessed by richness Molecular methods (inverse Simpson and ACE index) and diversity (Shannon Samples were sent to the Australian Genome Research and Simpson index). Facility in Urrbrae, Adelaide Australia. DNA was extracted using the DNeasy PowerSoil Pro DNA Extrac- tion Kit (Qiagen, Venlo, the Netherlands) according to Results the manufacturer′s instructions. A total of 79 whole tick Tick species specimens were then sequenced on an Illumina MiSeq A total of 447 tick specimens were collected from 47 platform [36]. Based on previous studies [37], the pres- bare-nosed wombats in New South Wales (NSW) and ence of bacteria in tick samples was detected using the Tasmania between January 2019 and January 2020 primer pair 341F (5′-CCT​AYG​GGRBGCASCAG-3′) and (Table 1). Five species of ticks comprising three genera 806R (5′-GGA​CTA​CNNGGG​TAT​CTAAT-3′) to amplify were morphologically identifed (Table 2); wombat tick (n Beard et al. Parasites Vectors (2021) 14:60 Page 4 of 18

Fig. 1 Geographic distribution of ticks collected from bare-nosed wombat (Vombatus ursinus) hosts between January 2019 and January 2020. Each point represents a unique collection location for the corresponding tick species. Overlapping points were displaced with a point displacement renderer around a centre symbol (denoted in the legend); point displacement distance was defned by number of map units (kilometres)

autumn (6%) and summer (6%); the remaining ticks were = 420; Fig. 2d), wallaby tick (n = 8; Fig. 2f), bush tick (n older specimens for which only the year of collection was = 3; Fig. 2a, e), Australian paralysis tick (n = 4; Fig. 2b), recorded. and I. tasmani (n = 12; Fig. 2c). Approximate tick infesta- tion ranged from one to 73 ticks per wombat with a total mean infestation of 9.8 ± 3.9 ticks per host. Juvenile (joey at foot) wombats exhibited higher mean infestation rates NGS analysis and bacterial composition of wombat ticks A total of 5,890,950 bacterial sequences and 1,759 OTUs (25.3 ± 20.4), followed by adult female wombats (7.1 (average length 414.3 bases) were assigned; however, only ± 4.5) and adult male wombats (6.6 ± 3.6). Te wom- bat tick was the most prevalent tick species comprising 745 OTUs had greater than 100 total sequences from all 94% of the total number of samples and was present on tick samples. Ticks had an average of 74,569 assigned 97.9% (46/47) of wombat hosts. Approximate tick diver- sequences each (males 63,397 sequences, females 92,827 sity ranged from one to four tick species per wombat. Te sequences, nymphs 56,470 sequences and larvae 57,701 highest tick diversity was from an adult male wombat in sequences). Engorged females had an average of 99,550 Coolagolite in NSW, an adult male wombat from Dalgety assigned sequences in comparison to unfed females, NSW and a wombat of unknown age and sex at Quaama which had an average of 40,723 sequences. Te clos- NSW with three tick species identifed for each. Females est matches for bacterial isolates as determined through GenBank for taxa of interest are shown in Table 3. Pro- were the most abundant instar identifed (n = 164), fol- teobacteria comprised the majority of the bacterial phyla lowed by males (n = 129), nymphs (n = 115), and larvae composition (55.1%) followed by Firmicutes (21.9%) and (n = 39). Te majority of females (89%), nymphs (96.5%) and larvae (100%) were engorged or semi-engorged from Actinobacteria (18.4%), as shown in Fig. 4. At the genus a meal (Fig. 3). Larvae could be identifed to genus level Coxiella comprised 40.3% of the total composition level only. In addition to ticks, there were also inciden- followed by Staphylococcus (13%). Coxiella was the most tal collections of nine unidentifed feas and six lice (all of dominant genus detected in larvae with a mean preva- the latter were identifed as Boopia tarsata). Most ticks lence of 81.6%. Nymphs were less likely to be infected were collected in winter (58%), followed by spring (25%), with one dominant phyla of bacteria than other instars Beard et al. Parasites Vectors (2021) 14:60 Page 5 of 18

Table 1 Study population of bare-nosed wombats (Vombatus ursinus) used for tick collection in this study Collection location GPS coordinates No. of hosts No. of ticks

Cedar Creek, NSW 32°49′30.32″S, 151°9′2.23″E 7 (6 ♂, 1 ♀) 32 (17 ♂, 15 ♀) Rock Flat, NSW 36°25′34.0284″S, 149°11′2.7132″E 2 (1 ♂, 1 U) 18 (2 N, 16 ♀) Bells Line of Road, NSW 33°31′1.0272″S, 150°28′47.316″E 1 ♀ 5 (2 ♂, 3 ♀) Murrabrine Forest Road, Yowrie, NSW 36°20′42.576″S, 149°45′33.12″E 2 ♂ 9 (1 N, 1 ♂, 7 ♀) Bridge over Colombo Creek, Bemboka, NSW 36°38′8.9052″S, 149°34′38.1792″E 1 ♀ 2 ♀ Rilys Road, Coolagolite, NSW 36°22′58.6416″S, 150°0′53.91″E 2 (1 ♂, 1 ♀) 4 ♀ Wolgan Valley, NSW 33°13′42.978″S, 150°11′10.2948″E 2 U 6 (1 L, 5 N) Wagga Wagga, NSW 35°6′54.6696″S, 147°22′32.5344″E 1 ♂ 1 ♂ The Rock, NSW 35°16′5.1528″S, 147°6′43.668″E 1 U 1 ♂ Werombi Road, Orangeville, NSW 34°1′23.8728″S, 150°39′22.7088″E 1 U 6 (4 N, 2 ♀) West Parade, Thirlmere, NSW 34°13′16.9932″S, 150°33′26.55″E 1 U 10 (4 N, 3 ♂, 3 ♀) West Parade, Couridjah, NSW 34°13′38.6472″S, 150°33′11.124″E 1 U 9 (7 N, 2 ♀) Picton, NSW 34°10′9.2856″S, 150°36′32.5008″E 1 U 41 (28 N, 7 ♂, 6 ♀) Spring Creek Road, Mount Hunter, NSW 34°4′53.976″S, 150°37′46.2108″E 1 ♂ 5 (4 ♂, 1 ♀) Eastview Drive, Orangeville NSW 34°0′54.0756″S, 150°35′11.9508″E 1 ♂ (J) 16 (6 ♂, 10 ♀) Silverdale Road, The Oaks, NSW 34°4′8.1624″S, 150°34′25.6656″E 1 ♀ 10 (5 ♂, 5 ♀) Moulders Road, Orangeville, NSW 34°2′44.4804″S, 150°34′23.4732″E 1 ♀ 11 ♀ Couridjah, NSW 34°13′54.8832″S, 150°32′58.0308″E 1 ♀ 9 (2 ♂, 7 ♀) Pheasants Nest Road, Pheasant Nest, NSW 34°15′15.318″S, 150°37′47.9784″E 1 ♂ (P) 2 N Mowbray Park Road, Mowbray Park, NSW 34°9′39.51″S, 150°32′54.1428″E 1 ♂ 4 (1 ♂, 3 ♀) Buxton Road, Buxton, NSW 34°15′4.5108″S, 150°31′34.1688″E 1 ♀ 26 (2 N, 21 ♂, 3 ♀) Kangaroo Valley, NSW 34°44′31.7436″S, 150°33′8.028″E 1 U (J) 73 (32 N, 15 ♂, 26 ♀) Bellmount Forest, NSW 34°54′14.4612″S, 149°14′54.0888″E 1 ♀ 4 (1 ♂, 3 N) Bellmount Forest, NSW 34°53′58.7832″S, 149°14′53.2392″E 1 ♂ 20 (3 N, 15 ♂, 2 ♀) Holbrook Road, Gelston Park, NSW 34°13′33.9996″S, 147°20′14.3088″E 1 ♂ 37 ♂ Rilys Road, Coolagolite, NSW 36°22′58.7244″S, 150°0′54.162″E 2 ♂ 13 (3 N, 1 ♂, 9 ♀) Captains Flat Road, Primrose Valley, NSW 35°27′14.8644″S, 149°25′8.0544″E 1 ♂ 3 (1 ♂, 2 ♀) Hard Road, Burra, NSW 35°33′28.4436″S, 149°13′19.3296″E 1 ♀ (J) 44 (38 L, 5 N, 1 ♂) Ironmungie Road, Dalgety NSW 36°33′53.6148″S, 148°55′7.5288″E 1 ♂ 7 ♀ Gidleigh Lane, Bungendore, NSW 35°17′43.656″S, 149°27′21.3192″E 1 ♀ (J) 14 N U U 1 U 10 (2 N, 8 ♀) Cradle Mountain Road, Cradle Mountain, Tasmania 41°31′23.2716″S, 146°4′32.6388″E 1 ♀ (J) 3 (1 N, 2 ♀) NSW New South Wales, GPS Global Positioning System, U unknown, L larvae, N , P pinky (unfurred joey), J joey

Table 2 List of the tick species collected and identifed from bare-nosed wombat (V. ursinus) hosts between January 2019 and January 2020 Tick species Common name No. collected Instar Locality

Bothriocroton auruginans Wombat tick 420 128 ♂, 141 NSW: Coolagolite, Rock Flat, Yowrie, Bellmount Forest, Bilpin, Bem- ♀, 112 N, boka, Buxton, Primrose Valley, Courijah, Orangeville, Bungendore, 39 L Burra, Gelstone Park, Dalgety, Kangaroo Valley, Mowbray Park, Pheasant Nest, Picton, Quaama, The Oaks, Mount Hunter, The Rock, Wagga Wagga, Thirlmere, Wolgan Valley Haemaphysalis bancrofti Wallaby tick 8 8 ♀, 2 N NSW: Coolagolite, Dalgety, Picton, Quaama Haemaphysalis longicornis Bush tick 3 1 ♂, 2 ♀ NSW: Coolagolite Ixodes tasmani Common marsupial tick 12 11 ♀, 1 N NSW: Dalgety. Tasmania: Cradle Mountain Ixodes holocyclus Australian paralysis tick 4 4 ♀ NSW: Coolagolite, Quaama For abbreviations, see Table 1 Beard et al. Parasites Vectors (2021) 14:60 Page 6 of 18

Fig. 2 a Bush tick Haemaphysalis longicornis female (i) dorsal, (ii) ventral; b Australian paralysis tick Ixodes holocyclus female (i) dorsal, (ii) ventral; c common marsupial tick Ixodes tasmani female (i) dorsal, (ii) ventral; d wombat tick Bothriocroton auruginans female (i) dorsal, (ii) ventral; e bush tick Haemaphysalis longicornis male (i) dorsal, (ii) ventral; f wallaby tick Haemaphysalis bancrofti female (i) dorsal, (ii) ventral

and often exhibited equal frequencies of three phyla. in 72% of B. auruginans (86% of females, 68% of males, Male and female adult ticks were predominantly associ- 39% of nymphs and 100% of larvae) but not detected in I. ated with Proteobacteria (Table 4). tasmani. Females had a mean prevalence of 51.7%, males Four OTUs (OTU_1, LC464975, 99% identity; 30.7%, nymphs 19.6% and larvae 82.3% for C. burnetii. OTU_977, LC464975, 94.41% identity; OTU_1383, Te distribution of C. burnetii-infected ticks detected in LC464975, 98.51% identity; and OTU_1806, CP014561, this study is shown in Fig. 5. 93.26% identity) were identifed as a species of Coxiella OTU_9 was assigned to a Rickettsiella endosymbiont closest matched to Coxiella burnetii and were detected of the common marsupial tick (KP994859, 100% identity) Beard et al. Parasites Vectors (2021) 14:60 Page 7 of 18

Frequency of tick species and tick instars collected from bare-nosed wombats (Vombatus ursinus)

20 tickinstar Female (engorged) Female (semi-engorged)

10 01 Female (unfed) Larvae Male Nymph (engorged) 80 Nymph (semi-engorged) Nymph (unfed) y c n e u 60 q e r F 40 20 0

Bothricroton auruginans Haemaphysalis bancrofti Haemaphysalis longicornis Ixodes holocyclus Ixodes tasmani

Tick Species Fig. 3 Species of tick and frequency of each instar collected from bare-nosed wombat (V. ursinus) hosts between January 2019 and January 2020

and comprised 94.5% of the bacterial diversity in the very high sequence numbers in four female B. aurugi- single female I. tasmani sample. Tis tick was collected nans collected in Orangeville NSW. Nine B. auruginans from a wombat in Dalgety NSW, which is 100 km from (two females, three males, three nymphs and one lar- the collection location of the wombat in Coolangubra vae) had Streptococcus salivarius (OTU_51, MN559932 NSW from which this sequence was originally isolated 100% identity), and Streptococcus didelphis (OTU_1504, [6]. OTU_79 was assigned to Candidatus Borrelia ivo- NR_115730, 99.53% identity) was detected in a low num- rensis (KT364340, 99.53% identity) and was detected ber of sequences (< 200) in one female and one nymph of in only one engorged adult female B. auruginans (2051 B. auruginans. sequences) from Mowbray Park in NSW. An uncultured (OTU_4, NZ_CP045277, 100% iden- Anaplasma sp. (OTU_29, MK041546, 98.51% identity) tity) was identifed in 21% of B. auruginans ticks (14% was detected in four female B. auruginans from Quaama, of females, 50% of nymphs, 50% of larvae and no males) Coolagolite and Te Oaks NSW. but not in I. tasmani. OTUs that had a taxonomic iden- Te genus Staphylococcus was identifed in six OTUs tity associated with environmental bacteria such as and was present in 66% of samples. OTU_14 (MT214233, Acidobacteria, Bacteroidetes and Cyanobacteria com- 100% identity) was assigned to Staphylococcus sciuri prised <4% of the total composition. Skin and soil-asso- and was present in 21% of B. auruginans samples (29% ciated bacteria that occurred in high sequence numbers of females, 27% of males, no nymph or larvae) but not included Corynebacterium ulcerans (OTU_20, 100% in I. tasmani. OTU_2 (MN314593, 100% identity), identity), Corynebacterium amycolatum (OTU_6, OTU_1817 (MN314593, 96.50% identity) and OTU_1923 MK465377, 100% identity), brunensis (MN314593, 94.87% identity) had a top BLAST hit of (OTU_8, MK097326, 100% identity), Comamonas serini- Staphylococcus agnetis and were present in 56% of sam- vorans (OTU_11, 9 CP021455, 9.77% identity), Parabur- ples (60% of females, 41% of males, 56% of nymphs and kholderia cafeinilytica (OTU_17, MN150516, 100% 75% of larvae) including the common marsupial tick. identity), and Dietzia timorensis (OTU_43, MN511783 Two additional OTUs were assigned to miscellaneous 100% identity). Staphylococcus spp. (OTU_15, MH549514, 100% iden- tity; and OTU_1791, MG572712, 99.53% identity) but Discussion were represented in very low numbers of sequences. Tis study aimed to record the species of ticks that feed Eight OTUs were assigned to the genus Streptococ- on bare-nosed wombats and identify the bacteria asso- cus; however, only three were present in more than 100 ciated with them. Five tick species were collected and sequences in any of the ticks. Streptococcus dysgalac- included the frst record of H. longicornis on bare-nosed tiae (OTU_5, CP044102, 100% identity) was detected in wombats. A very high number of bacterial sequences Beard et al. Parasites Vectors (2021) 14:60 Page 8 of 18

Table 3 Bacterial composition of ticks parasitising bare-nosed wombat (V. ursinus) hosts between January 2019 and January 2020 Tick species Locality Closest match in GenBank (% similarity) No. positive Length (bp) Bit-score

B. auruginans (wombat tick) NSW Coxiella burnetii (99.07%) 56/78 429 771 Staphylococcus sciuri (100%) 16/78 429 793 Corynebacterium amycolatum (100%) 9/78 410 758 Dermacoccus nishinomiyaensis (97.80%) 12/78 409 706 Macrococcus brunensis (100%) 20/78 429 793 Planomicrobium glaciei (100%) 4/78 428 791 Lysinibacillus sp. (100%) 8/78 426 787 Brachybacterium paraconglomeratum (100%) 13/78 409 756 Escherichia coli (100%) 16/78 429 793 Acinetobacter sp. (100%) 19/78 430 795 Pseudomonas sp. (100%) 7/78 429 793 Candidatus Borrelia ivorensis (99.53) 1/78 424 784 Uncultured Anaplasma sp. (98.51%) 4/78 404 713 I. tasmani (common marsupial tick) NSW Rickettsiella endosymbiont (100%) 1/1 429 793

Only taxa of interest are shown, and numbers of positive samples are based on samples with > 100 assigned operational taxonomic units (OTUs) bp Base pair were detected in wombat ticks, highlighting the efec- is an introduced three-host tick distributed from south- tiveness of NGS and the diversity of microorganisms in east Queensland to Victoria [52]. A new tick record for Australian ticks. Proteobacteria, Firmicutes and Actino- bare-nosed wombats, the bush tick collected in this bacteria dominated the bacterial profle, and the bacterial study was positively diferentiated from similar species composition of the ticks studied supports similar investi- by 5+5 dentition and sharply pointed spurs on coxa gations into these species [14, 17, 29]. 1 [30]. Te adult male bush tick found in Coolagolite Te wombat tick B. auruginans is consistently the NSW is particularly unusual considering this species most prevalent tick found on bare-nosed wombats is an obligate parthenogen in Australia, resulting in [46–48], and all instars except larvae were represented males being quite rare [53]. , sheep and horses in this study. All larval specimens collected were identi- are the preferred hosts for this species, but it has also fed as Bothriocroton sp. and shared their host with only been collected from humans, domestic animals, various B. auruginans instars. It is likely that these larval speci- species of , black-striped wallabies (Wallabia dor- mens were B. auruginans due to host specifcity of other salis), northern brown (Isoodon macrourus) Bothriocroton spp.; however, this could not be confrmed. and common wallaroos (Macropus robustus) [30, 31]. Heavy has been associated with anae- Te three specimens collected in this study were from mia and poorer health parameters in other native mar- a free-ranging wombat on a 100-acre property with supials [49, 50], and at least two of the wombats in this no active livestock; however, access to properties with study were diagnosed with anaemia as a result of their livestock is possible across dried creek beds at certain tick burden (D. Kerr, personal communication). While it times of the year (D. Ondinea, personal communica- has been suggested that B. auruginans occurs throughout tion). Te bush tick has been extensively studied over- most of the bare-nosed wombat range in NSW [51], the seas and is considered a vector of bacteria, and only confrmed localities in the state are Burrawang [46], , in particular C. burnetii [54], Ehrlichia chaf- Tooloom, Armidale [30] and Wee Jasper [17]. Tis study feensis, Borrelia spp. [55], and Teileria orientalis [56]; provides additional locality reports for B. auruginans and however, transmission has not been shown to occur in highlights the abundance of this species on bare-nosed Australian specimens [57]. wombats. Despite the host specifcity of B. auruginans, it Te Australian paralysis tick is well known for caus- has been suggested that it is likely a three-host tick like ing in domestic animals and humans [58]. other Bothriocroton sp., which parasitize reptiles [30]; Native Australian marsupials and eutherians have, how- however, further research on the life cycle and seasonal- ever, co-evolved with the Australian paralysis tick, are ity of this species is needed. the natural hosts for this tick and are typically immune Known as the bush tick in Australia and the cattle to tick paralysis [59]. Found along the entire east coast tick or Asian longhorned tick elsewhere, H. longicornis of Australia, the Australian paralysis tick is an eclectic Beard et al. Parasites Vectors (2021) 14:60 Page 9 of 18

Fig. 4 Taxonomic summary of bacterial phyla found in wombat ticks between January 2019 and January 2020 Beard et al. Parasites Vectors (2021) 14:60 Page 10 of 18

Table 4 List of wombat tick samples sequenced on the Illumina MiSeq platform and absolute OTU counts for each sample Sample code Species Common name Sex Instar Total abundance Total abundance Total C. burnetii Staphylococcus agnetis abundance Rickettsiella

39a B. auruginans Wombat tick Female Adult 96,850 240 0 39b B. auruginans Wombat tick Female Adult 38,832 617 0 40a B. auruginans Wombat tick Male Adult 424 86 0 40b B. auruginans Wombat tick Male Adult 499 37 0 40c B. auruginans Wombat tick Male Adult 0 52 0 40d B. auruginans Wombat tick Male Adult 245 33 0 41a B. auruginans Wombat tick Female Adult 118,871 23 0 41b B. auruginans Wombat tick Female Adult 135,810 1 0 41c B. auruginans Wombat tick Female Adult 151,567 0 0 41d B. auruginans Wombat tick Female Adult 35,440 0 0 42a B. auruginans Wombat tick Female Adult 121,194 0 0 42b B. auruginans Wombat tick Male Adult 11,701 0 0 43a B. auruginans Wombat tick Female Adult 42 49 0 44a B. auruginans Wombat tick Female Adult 22,187 3 0 44b B. auruginans Wombat tick Female Adult 150,092 74 0 46a B. auruginans Wombat tick Female Adult 8363 0 0 47b B. auruginans Wombat tick Female Adult 0 0 0 48a B. auruginans Wombat tick - Nymph 7320 0 0 48b B. auruginans Wombat tick Female Adult 694 0 0 49a B. auruginans Wombat tick - Nymph 56,494 0 0 49b B. auruginans Wombat tick Male Adult 82,607 155 0 49c B. auruginans Wombat tick Male Adult 28,893 51,956 0 49d B. auruginans Wombat tick - Nymph 0 0 0 49f B. auruginans Wombat tick Male Adult 0 1158 0 50a B. auruginans Wombat tick Female Adult 87,091 160 0 51a B. auruginans Wombat tick Female Adult 6965 328 54 51b B. auruginans Wombat tick Female Adult 172 100 0 51c B. auruginans Wombat tick Male Adult 0 194 0 51d B. auruginans Wombat tick Male Adult 0 293 69 52a B. auruginans Wombat tick Female Adult 47,707 1719 0 52b B. auruginans Wombat tick Female Adult 36,035 1682 0 52c B. auruginans Wombat tick Male Adult 107,270 195 0 52d B. auruginans Wombat tick Male Adult 47,213 432 0 53a B. auruginans Wombat tick Female Adult 12,669 520 0 53b B. auruginans Wombat tick Female Adult 3819 1029 0 53c B. auruginans Wombat tick Female Adult 2535 1130 0 53d B. auruginans Wombat tick Female Adult 2314 3475 0 54a B. auruginans Wombat tick Female Adult 47 2612 0 54c B. auruginans Wombat tick Female Adult 6442 137 0 55a B. auruginans Wombat tick Female Adult 76,233 6706 0 55b B. auruginans Wombat tick Male Adult 4771 119 0 56a B. auruginans Wombat tick Male Adult 72,344 0 0 56b B. auruginans Wombat tick Male Adult 0 0 0 56d B. auruginans Wombat tick Male Adult 20,493 0 0 56e B. auruginans Wombat tick Male Adult 2468 0 0 56f B. auruginans Wombat tick Male Adult 0 0 0 57a B. auruginans Wombat tick - Nymph 0 39,411 0 57b B. auruginans Wombat tick - Nymph 2 106,880 0 Beard et al. Parasites Vectors (2021) 14:60 Page 11 of 18

Table 4 (continued) Sample code Species Common name Sex Instar Total abundance Total abundance Total C. burnetii Staphylococcus agnetis abundance Rickettsiella

57c B. auruginans Wombat tick - Nymph 0 5956 0 57d B. auruginans Wombat tick - Nymph 0 5700 0 57e B. auruginans Wombat tick - Nymph 0 12,343 0 57f B. auruginans Wombat tick - Nymph 0 9720 0 58a B. auruginans Wombat tick Male Adult 107,406 44 0 58b B. auruginans Wombat tick Male Adult 1859 348 0 58c B. auruginans Wombat tick Male Adult 9 0 0 58d B. auruginans Wombat tick - Nymph 449 0 0 58e B. auruginans Wombat tick - Nymph 9918 0 0 58f B. auruginans Wombat tick - Nymph 8 0 0 59b B. auruginans Wombat tick - Nymph 93,668 0 0 60a B. auruginans Wombat tick Male Adult 8398 1543 0 60c B. auruginans Wombat tick Male Adult 128,271 30 0 60d B. auruginans Wombat tick Male Adult 92,622 99 0 60e B. auruginans Wombat tick Male Adult 99,967 3503 0 60f B. auruginans Wombat tick Male Adult 129,252 5470 0 60g B. auruginans Wombat tick Male Adult 55,074 903 0 61c B. auruginans Wombat tick - Nymph 1709 1 0 62a B. auruginans Wombat tick Female Adult 131,882 1 0 63a B. auruginans Wombat tick Male Adult 101 3536 0 64b B. auruginans Wombat tick - Larvae 37,394 1684 0 64c B. auruginans Wombat tick - Larvae 61,504 14,953 0 64d B. auruginans Wombat tick - Larvae 61,897 466 0 64e B. auruginans Wombat tick - Larvae 27,290 0 0 65a B. auruginans Wombat tick Female Adult 147,715 143 14 65b B. auruginans Wombat tick Female Adult 0 1453 1 66a B. auruginans Wombat tick - Nymph 118,704 969 0 66c B. auruginans Wombat tick - Nymph 0 17,181 0 66d B. auruginans Wombat tick - Nymph 0 3741 0 67b B. auruginans Wombat tick - Nymph 0 6881 0 67c Ixodes tasmani Common marsupial tick Female Adult 0 173 85,653 feeder and has been found on many diferent and species in Australia and has been associated with various species; however, in certain areas it is depend- pathogens such as Rickettsia, Rickettsiella, , ent upon bandicoots to survive between seasons [30]. All Teileria, nematodes and Hepatozoon [29, 61–66]. Regu- specimens collected in this study were engorged females; larly found on bare-nosed wombats in low numbers [48, however, adult males are rarely seen, as mating occurs of 67], I. tasmani is a nidicolous species that detaches from the host and adult male ticks feed on adult female ticks its nocturnal vertebrate hosts during the day and is there- as opposed to the mammalian hosts [60]. With the use fore likely associated with wombat burrows. Given its of targeted blocking primers a Borrelia sp. fast reproductive rate, three-host life cycle and the vari- was recently isolated from a single Australian paralysis ety of pathogenic organisms that it typically harbours, tick collected from an echidna [13], highlighting the hid- this species is likely to pose a disease threat to wombats den pathogenic potential of this species. and wombat handlers; however, more research needs to Like the Australian paralysis tick, the common marsu- be conducted to determine the extent of this threat. pial tick is similarly indiscriminate in its feeding habits An endemic tick that primarily feeds on macropods, having been found on various wildlife, domestic animals wallaby tick is predominantly distributed through- and humans. However, it is the most widespread Ixodes out coastal Queensland and northern NSW, and apart Beard et al. Parasites Vectors (2021) 14:60 Page 12 of 18

Fig. 5 Geographic distribution of Coxiella burnetii detected in ticks from bare-nosed wombat (V. ursinus) hosts between January 2019 and January 2020. Each point corresponds with the collection location of the tick(s) which were positive (> 100 sequences) for C. burnetii. A point cluster renderer was used to group nearby points into a single rendered marker symbol. Point cluster distance was determined by point units

from a disjunct population on Raymond Island Victo- that for some species of native ticks, including the ria, the southernmost reports of this species are from wombat tick, a larger number of sequences is required a bare-nosed wombat, a red-necked wallaby (Macropus to produce an accurate representation of bacterial rufogriseus) and a swamp wallaby (Wallabia bicolor) diversity [17]. Te most abundant and diverse phylum in the Nadgee State Forest NSW [24]. Te specimens was the Proteobacteria, which is consistent with similar collected in the present study were from Dalgety and studies of native hard ticks [29, 70]. Quaama NSW, which are located approximately 2 h Pathogens previously isolated from B. auruginans north of Nadgee. Tese new specimens further con- include C. burnetii, and Rickettsia frm the presence of wallaby tick in the far south NSW typhi [29] and varying levels of Proteobacteria and Fir- region and provide the second account of this species micutes [14, 17]. Te very high prevalence of C. bur- feeding on bare-nosed wombats [24]. Although macro- netii found in all B. auruginans instars in this study is pods are the native host for H. bancrofti, there are more similar to previous fndings in this species [29]. Cox- records of this species from cattle than native animals iella-like organisms are known to be highly efcient at [24], and it is one of the main vectors of T. orientalis transovarial transmission between tick hosts [71], and that impacts cattle in Australia [57, 68]. their presence within Malpighian tubules may sug- Analysis of alpha-diversity (Fig. 6) showed high vari- gest that they play a role in tick nutrition [7]. Difer- ability across both sample locations and instars, similar ent strains of C. burnetii have been shown to be highly to previous studies [17, 69]. However, there was some related (> 99%) based on 16S rRNA, highlighting that similarity between the same instars from the same col- the species recently evolved from an ancestral symbi- lection location. Diversity can vary greatly between tick ont of ticks [6]. Because B. auruginans exhibits such studies depending on extraction methods, the quality of remarkable host specifcity, it is unlikely that this spe- fltering and bioinformatic analysis. All samples in this cies is a signifcant vector for C. burnetii in humans. It study underwent identical extraction, library prepara- is unknown, however, what impact this pathogen has tion and bioinformatic analysis so it is possible that this on both healthy and sarcoptic mange-afected wom- afected sequencing depth of samples. It has been noted bats. Blood and urine samples taken from wombats Beard et al. Parasites Vectors (2021) 14:60 Page 13 of 18

have failed to indicate the presence of C. burnetii [72], species such as Staphylococcus aureus are associated with whereas other native marsupials such as the , ban- Sarcoptes scabiei and responsible for causing sca- dicoots and macropods are regularly found to be sero- bies-associated pyoderma in humans [86]. Two species positive for this bacterium [73–75]. Further studies to of Staphylococcus were detected in this study: S. agnetis, investigate the presence of C. burnetii in wombat faeces which is typically associated with clinical disease in cattle and blood, and in parasites other than B. auruginans, and poultry [87, 88], and S. sciuri, which is a skin-associ- may be benefcial to determine the importance, role ated bacterium acquired through contact with host skin and impact of this pathogen in wombats and wombat [84]. Staphylococcus sciuri has also been detected in feas ticks. from bandicoots and in Australia, and in various lice Te presence of Borrelia in Australian ticks is a recent and tick species including I. holocyclus and H. longicornis discovery [13], and targeted approaches using blocking [85]. primers and highly conserved housekeeping genes have Further skin-related bacteria found included C. ulcer- provided insights into potential reservoirs and vectors of ans, which causes a zoonotic similar to diphthe- novel Borrelia sp. in Australia [14]. A species of Borrelia ria [88], Dolosigranulum pigrum, which is associated with had the closest match to Candidatus Borrelia ivorensis in humans [89, 90], and Macrococcus brunen- and was detected in a single Bothriocroton auruginans sis, which is phylogenetically similar to a species of Mac- from NSW. Te original isolate for this species was from rococcus responsible for causing skin infection in dogs variegatum in western Africa, and it is [91]. At least three distinct species of Streptococcus were more closely related to the relapsing fever Borrelia group detected, of which S. dysgalactiae and S. didelphis are than the Lyme group [76]. Te uncultured Anaplasma important pathogens of humans and animals causing skin sp. detected was originally isolated from an echidna tick infection [92, 93]. Other species of Streptococcus such as (Bothriocroton concolor). All recognised Anaplasma spp. Streptococcus pyogenes from Sarcoptes scabiei mites are are obligate intracellular tick-borne mammalian patho- responsible for causing skin infection in humans [86]. gens [77], and as transovarial transmission between ticks Te pathogenicity and consequences of these skin-asso- has not yet been shown, it is believed that this genus per- ciated bacteria on both healthy and sarcoptic mange- sists solely through infected mammalian hosts [78]. impacted wombats may therefore be important. A commensal bacterium of the mammalian gastro- Endosymbiotic bacteria are an important compo- intestinal tract, E. coli is commonly found in native nent of the tick microbiome and often play a role in tick , with the highest prevalence in herbivorous reproductive and nutritional ftness [15]. Te tick endo- mammals with larger body masses [79]. Some species of symbionts found in this study include Rickettsiella, Aci- E. coli are zoonotic and impact human health [80]. One netobacter and Pseudomonas. Te genera Acinetobacter study found northern hairy-nosed wombats (Lasiorhinus and Pseudomonas have previously been isolated from kreftii) to have an E. coli prevalence of 80% and southern wombat feas [85]; they are also found in all Ixodes exam- hairy-nosed wombats (Lasiorhinus latifrons) to have 86% ined, and are believed to play an important role in the [79]; however, another study found no zoonotic E. coli physiological processes of ticks [93]. Despite I. tasmani in all three species of wombats [81]. While a strain of E. exhibiting a very high prevalence of a Rickettsiella endo- coli occurs in B. auruginans, it has been shown that ticks symbiont, some known tick endosymbionts such as Wol- exhibit various innate immune responses to this bacte- bachia and Francisella were not detected in this study. rium [82, 83] and it is destroyed in the body of the tick It is believed that bacterial endosymbionts are domi- rather than harboured and transmitted. nant in the majority of ixodid ticks [9], and there are Ticks are often found to have large quantities of bac- examples of endosymbiotic bacteria so abundant they teria that are associated with the soil environments in mask other microbes including pathogens, for example which they spend most of their lives, in addition to bacte- Candidatus Midichloria mitochondrii in the Austral- ria associated with the skin of their mammalian hosts [13, ian paralysis tick [13]. DNA extracted from whole tick 84]. Some pathogenic environmental and skin-associated specimens, in particular those which have fed from their bacteria that were detected in both the wombat tick and vertebrate hosts, will contain tick DNA, host DNA and common marsupial tick may have potential implications microbial DNA (i.e. bacterial, viral, eukaryotic). Te pres- for wombats with sarcoptic mange, or could even have ence of host DNA in engorged ticks has been known to been detected as a result of the ticks feeding on wombats cause difculties due to inhibitory properties in mam- with sarcoptic mange-associated bacteria. malian blood [94], so a targeted approach is required Members of the genus Staphylococcus are typically when examining bacterial communities. Popular genetic commensals of mammalian skin, and are commonly markers used for molecular identifcation of ticks and found in ticks of native Australian wildlife [84, 85]; some their associated bacteria, include the cytochrome c Beard et al. Parasites Vectors (2021) 14:60 Page 14 of 18

Fig. 6 Alpha-diversity of bacterial composition in ticks collected from bare-nosed wombats (V. ursinus) between January 2019 and January 2020 assessed by diversity (Shannon, Simpson) and richness (ACE, inverse Simpson) Beard et al. Parasites Vectors (2021) 14:60 Page 15 of 18

oxidase subunit 1 (COI) protein-coding gene, and the highlights the high bacterial diversity in native Austral- 16S rRNA, 12S rRNA and 18S rRNA genes [94]. Each has ian ticks that was unrecognised prior to the development its advantages and limitations, for example COI ofers of NGS. Furthermore, the detection of C. burnetii in a an extensive existing library of universal primers as it large proportion of wombat ticks highlights the need for is the standard marker for barcoding of animal species; further investigation into wombat ectoparasites and their however, it is limited in its ability to distinguish certain associated pathogens, in addition to the ability of wom- groups of organisms such as the Ixodidae to species level. bats to cope with these pathogens and tick burdens in the Te 16S rRNA gene is the most commonly used molecu- presence of sarcoptic mange. Te complex and dynamic lar marker because it can accurately distinguish between relationships between vertebrate wildlife hosts, ticks and most prokaryotic taxa, but some microbial groups such pathogens are continuously highlighted in the northern as may be difcult to distinguish due to their hemisphere [96, 97]. Te unique evolutionary history of interspecifc 16S rRNA similarity [95]. Tere are nine Australian fauna and tick species is shown in the distinct hypervariable regions of bacterial 16S rRNA genes that diversity yet taxonomic diferences of these tick-host- can be efectively targeted to identify bacterial taxa (V1- pathogen relationships from those overseas. With the V9), and regions V1-V4 have been most commonly used advancement of molecular methods the extent of these in ticks [15]. unique evolutionary relationships will become clearer, Te sampling method used in this study was both eco- and may lead to potential improvements in the manage- nomical and allowed for a fair assessment of tick infesta- ment of vector-borne diseases such as sarcoptic mange. tion rates on wombat hosts. However, it can be assumed that in some cases smaller nymphal and larval tick instars Abbreviations were likely overlooked. It is also likely that some ticks had NGS: Next-generation sequencing; OTU: Operational taxonomic unit; COI: left road-killed wombats which were opportunistically Cytochrome c oxidase subunit 1. sampled, despite Skerratt et al. [48] fnding no diference Acknowledgements between tick density on live or road-killed wombats. Te Thank you to Amanda Cox, Danie Ondinea, Bronlow Hall, Inga Schwaiger, high abundance of female instars is likely indicative of Belinda Goldsworthy, Lyn Oberon, Diane Hinton, Liesl Perryman, Elena Guar- racino, Pru Carpenter and Deborah Kerr for assisting with tick collection, and some collection bias due to their larger size. Te collec- Siobhon Egan for assisting with tick identifcation. Donations to Wombat tion of ticks from animals in care limits the assessment Giving available through the WomSAT website funded this project. of the origin of tick species and species of microorgan- Authors’ contributions isms due to the uncertainty of whether the ticks attached HJS and JMO: Conceptualized the study and provided supervision and valida- in the location of rehabilitation or the original habitat tion for the study. DB: Obtained the samples, analysed the data and wrote where the wombat was collected from. Tree species of the original draft of the manuscript. All authors read and approved the fnal manuscript. ticks collected from wombats (the wallaby, bush and Aus- tralian paralysis tick) could not be processed for bacte- Funding rial presence in this study. However, these tick species Western Sydney University, Wombat Giving. are known vectors of signifcant pathogens of domestic Availability of data and materials animals and humans, and as a result have been exten- The datasets used and/or analysed during the current study are available sively studied. All the ticks collected in this study except from the corresponding author on reasonable request. Voucher specimens have been submitted to the Australian Museum and include Bothricroton for the Australian paralysis tick were non-nidicolous hard auruginans KS.130891, Haemaphysalis bancrofti KS.130892, Ixodes holocyclus ticks and presumably picked up by the wombat hosts KS.130893, Ixodes holocyclus KS.130893, Haemaphysalis longicornis KS.130894 whilst they were grazing. Many of the wombats used in and Ixodes tasmani KS.130895. this study were also in an atypical environment and had Ethics approval and consent to participate not had recent exposure to burrows. Considering that The collection of invertebrates described here did not require ethics approval. the majority of soft ticks are nidicolous and feed for Consent for publication very short periods of time, further investigation into the Not applicable. ticks associated with wombat burrows would provide a broader perspective of all the tick species associated with Competing interests The authors declare that they have no competing interests. wombats. Author details Conclusions 1 School of Science, Western Sydney University, Penrith, New South Wales, Australia. 2 School of Animal and Veterinary Sciences, Charles Sturt University, Tis study builds upon recent wildlife tick research and Wagga Wagga, NSW, Australia. provides the frst focused investigation into the ticks and tick-associated bacteria of bare-nosed wombats. Received: 1 May 2020 Accepted: 28 December 2020 Te detection of various Proteobacteria in this study Beard et al. Parasites Vectors (2021) 14:60 Page 16 of 18

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